Salivary glands produce secretions that bathe and protect the oral cavity. Cystatin S, a naturally occurring cysteine proteinase inhibitor secreted by submandibular glands, is believed to play a major protective role. Until recently, nothing was known about the regulation of cystatin, S gene expression despite the role these proteins play in oral pathophysiology. Our long-range goal is to determine the mechanisms regulating cystatin genes. The goal of the proposed research is to identify the functional regulatory elements of the rat cystatin S gene, highly homologous to human cystatin S genes, and the DNA binding proteins that modulate their function. The Specific aims are to: 1) examine the hormonal, neuronal, and developmental regulation of the rat cystatin S gene, 2) determine the functional cis-acting DNA regulatory elements in the cystatin S gene, and 3) identify, isolate and characterize the trans-acting DNA binding proteins regulating the rat cystatin S gene and the genes encoding them. We will examine cystatin S mRNA levels in the submandibular glands of postnatally developing males, of male and female rats after castration and hormone replacement, and in glands whose innervation was blocked. Transfection of segments of the 5' region of the cystatin S gene in constructs containing the CAT reporter gene into appropriate recipient cells will allow us to determine which of the potential regulatory sequences we have identified are functional. Gel shift and DNase I protection assays will permit us to identify the DNA binding proteins regulating this gene. Purifying these proteins, obtaining their amino acid sequence and synthesizing oligonucleotides corresponding to the amino acid sequence and generating antibodies to these DNA binding proteins will make it possible to clone and characterize the genes encoding these regulatory proteins. Knowledge of how expression of cystatin genes is regulated may lead to clinical treatments for patients who are orally compromised because of periodontal disease, Sjogren's syndrome, and HIV disease.